Photochemistry in cellular membranes is responsible for initiating many responses of cells to ultraviolet and visible radiation with and without added photosensitizing molecules. The plasma membrane and mitochondrial membranes are frequently the sites of primary photosensitization in the use of dyes plus visible radiation to treat tumors (photodynamic therapy, PDT). The long range objective of this application is to provide a rational basis for design of membrane-photosensitizing dyes for applications in medicine and biology. The specific aims of this proposal are: Specific Aim 1: To produce, separately, singlet oxygen (1O2*) and free radicals from the same photosensitizer molecules in the plasma membrane. Singlet oxygen will be produced using low intensity light; radicals will be produced using pulsed lasers. The ability of mathematical modeling to predict optimal laser intensity and wavelength for photosensitization of cells will be evaluated. Specific Aim 2: To establish the influence of the location and identity (1O2* or free radicals) of the primary reactive species formed during photosensitization on biochemical alterations produced in the plasma membrane. Photosensitizers will be used that localize at different sites in the plasma membrane or generate different reactive species. Changes in membrane functions will be quantitatively compared between photosensitizers. Membrane functions to be measured include proline uptake, activity of Na+,K+-ATPase, membrane permeability, intracellular Ca2+, and membrane potential. The kinetics for alteration of plasma membrane potential and intracellular Ca2+ will be monitored on a microsecond to minute time scale after laser excitation using fluorescent reporter dyes. Specific Aim 3: To determine whether cellular responses to membrane photosensitization reflect the identity or location of formation of the reactive species. The activation of the membrane-associated enzymes, phospholipases A2 and C, will be assessed after photosensitization with dyes producing either 1O2* or free radicals in the plasma membrane. Induction of early response genes will be used as used to measure the effects of reactive species on an early nuclear response to plasma membrane photosensitization. Measurements will include induction of c- jun, c-fos and c-myc mRNA. Induction of apoptosis by photosensitizers that cause plasma membrane damage will be assessed. After photosensitization, DNA will be isolated and cleavage into nucleosome- size fragments will be quantitated.